Our long term goal is to develop immune therapies for disease caused by Human Papillomavirus (HPV). While the field of cancer immunotherapy has shown proof of principle in humans, namely, that established malignancies can be recognized and eradicated by a tumor-specific adaptive T cell response, reproducibly achieving this effect has been difficult. One reason may be that therapeutic vaccines tested to date have not been immunogenic enough to eliminate established disease. Another reason may be immunologic suppression directly mediated by developing and progressing tumors, which are operative in the lesion microenvironment. This project will test the hypothesis that HPV-specific adaptive T cell responses can be elicited by therapeutic vaccination in subjects with high grade cervical dysplasia (CIN3), and that the application of a topical TLR7 agonist, imiquimod, directly on the lesion, will enhance access of CD8+ T cells to the lesions. CIN3 lesions are relatively accessible, and some of them do regress within our 15-week study window. The disease provides rational, non-self antigenic targets for therapeutic vaccination. This project will test a heterologous DNA prime, recombinant vaccinia-based boost regimen to enhance the immune response against HPV16 E6 and E7, which are expressed in a functionally obligate manner in cervical cancer and its precursor lesion, high grade cervical dysplasia (CIN3). This project also provides the opportunity to analyze target lesions directly to determine if we can identify evidence of mechanisms that would mitigate the function of immune effector responses, namely, impairment of homing and function of immune cell subsets. In fact, we found that CD8+ T cells accumulate at CIN3 lesions, but in the case of persistent disease, fail to access lesional epithelium. While patterns of immune cell infiltration have been identified to predict clinical outcome in the clinical setting of invasive disease, to our knowledge, they have not been identified early in disease, before development of an overtly invasive phenotype. This work is an opportunity to study immune responses that are localized to incipient neoplasia, and immune homeostatic mechanisms which could be locally uncoupled to enhance both access and function of effector immune cells to eliminate disease. RELEVANCE (See instructions); Cervical cancer is caused by human papillomavirus (HPV) infection. The goal of this research is to develop immune therapeutic strategies which could educate the immune response to target and eliminate established disease caused by HPV.